Acute megakaryoblastic leukemia in patients without Down syndrome is a rare malignancy with a poor prognosis. RNA sequencing of fourteen pediatric cases previously identified novel fusion transcripts ...that are predicted to be pathological including CBFA2T3-GLIS2, GATA2-HOXA9, MN1-FLI and NIPBL-HOXB9. In contrast to CBFA2T3-GLIS2, which is insufficient to induce leukemia, we demonstrate that the introduction of GATA2-HOXA9, MN1-FLI1 or NIPBL-HOXB9 into murine bone marrow induces overt disease in syngeneic transplant models. With the exception of MN1, full penetrance was not achieved through the introduction of fusion partner genes alone, suggesting that the chimeric transcripts possess a unique gain-of-function phenotype. Leukemias were found to exhibit elements of the megakaryocyte erythroid progenitor gene expression program, as well as unique leukemia-specific signatures that contribute to transformation. Comprehensive genomic analyses of resultant murine tumors revealed few cooperating mutations confirming the strength of the fusion genes and their role as pathological drivers. These models are critical for both the understanding of the biology of disease as well as providing a tool for the identification of effective therapeutic agents in preclinical studies.
In humans and in mouse models, precursor B-cell lymphoblastic leukemia (B-ALL)/lymphoblastic lymphoma (B-LBL) can be classified as either the pro-B or pre-B subtype. This is based on the expression ...of antigens associated with the pro-B and pre-B stages of B-cell development. Antigenic markers can be detected by flow cytometry or immunohistochemistry (IHC), but no comparison of results from these techniques has been reported for murine B-ALL/LBL. In our analysis of 30 cases induced by chemical or viral mutagenesis on a WT or Pax5+/– background, 18 (60%) were diagnosed as pro-B by both flow cytometry and IHC. Discordant results were found for 12 (40%); 6 were designated pro-B by IHC and pre-B by flow cytometry and the reverse for the remaining 6 cases. Discordance occurred because different markers were used to define the pro-B–to–pre-B transition by IHC vs flow cytometry. IHC expression of cytoplasmic IgM (μIgM) defined the pre-B stage, whereas the common practice of using CD25 as a surrogate marker in flow cytometry was employed here. These results show that CD25 and μIgM are not always concurrently expressed in B-ALL/LBL, in contrast to normal B-cell development. Therefore, when subtyping B-ALL/LBL in mice, an IHC panel of B220, PAX5, TdT, c-Kit/CD117, CD43, IgM, and ΚLC should be considered. For flow cytometry, cytoplasmic IgM may be an appropriate marker in conjunction with the surface markers B220, CD19, CD43, c-Kit/CD117, BP-1, and CD25.
Myeloid sarcoma is a rare condition consisting of extramedullary myeloid blasts found in association with acute myeloid leukemia or, in the absence of bone marrow involvement. We identified an infant ...with isolated myeloid sarcoma whose bone marrow was negative for involvement by flow cytometry. Sequencing revealed the fusion oncogene CIC-NUTM2A and identified the sarcoma to be clonally evolved from the bone marrow, which carried the fusion despite the absence of pathology. Murine modeling confirmed the ability of the fusion to transform hematopoietic cells and identified receptor tyrosine kinase (RTK) signaling activation consistent with disruption of the CIC transcriptional repressor. These findings extend the definition of CIC-rearranged malignancies to include hematologic disease, provide insight into the mechanism of oncogenesis, and demonstrate the importance of molecular analysis and tracking of bone marrow involvement over the course of treatment in myeloid sarcoma, including patients that lack flow cytometric evidence of leukemia at diagnosis.
This study illustrates molecular involvement of phenotypically normal bone marrow in myeloid sarcoma, which has significant implications in clinical care. Further, it extends the definition of CIC-rearrangements to include hematologic malignancies and shows evidence of RTK activation that may be exploited therapeutically in cancer(s) driven by these fusions.
Therapy‐related myeloid neoplasms (t‐MN) are a distinct subgroup of myeloid malignancies with a poor prognosis that include cases of therapy‐related myelodysplastic syndrome (t‐MDS), therapy‐related ...myeloproliferative neoplasms (t‐MPN) and therapy‐related acute myeloid leukemia (t‐AML). Here, we report a series of patients with clinical features consistent with juvenile myelomonocytic leukemia (JMML), an overlap syndrome of MDS and myeloproliferative neoplasms that developed after treatment for another malignancy.
Acute megakaryoblastic leukemia (AMKL) accounts for ~10% of childhood AML. AMKL patients without Down syndrome have a poor outcome with a 3 year survival of less than 40%. To gain insight into the ...biology of this disease, we previously performed transcriptome sequencing on diagnostic blasts from a discovery cohort of 14 pediatric cases and validated our findings in a recurrency/validation cohort consisting of 34 pediatric and 28 adult samples. This analysis identified novel fusion transcripts restricted to pediatric AMKL including CBFA2T3-GLIS2,GATA2-HOXA9, MN1-FLI1, and NIPBL-HOXB9.
To confirm their role in oncogenesis and gain insight into the mechanism whereby these fusions promote disease, we introduced each of them into murine hematopoietic cells and assessed their effect on in vitro colony replating as a surrogate measure of self-renewal. Hematopoietic cells transduced with a control retrovirus failed to form colonies after the second replating. By contrast, expression of each of the fusion genes resulted in a marked increase in self-renewal capacity, with colony formation persisting through 10 replatings. Immunophenotypic analysis revealed evidence of megakaryocytic differentiation in CBFA2T3-GLIS2 and MN1-FLI1 cohorts, whereas NIPBL-HOXB9 and GATA2-HOXA9 cells carried markers consistent with myeloid progenitors. Transplantation of fusion gene modified bone marrow cells into syngeneic recipients induced overt leukemia in all cohorts with the exception of CBFA2T3-GLIS2, suggesting an essential requirement for cooperative mutation(s) in cases expressing this chimeric gene. To assess self-renewal activity of the leukemia generated in our murine models, we conducted secondary transplants for all cohorts. In all cases, the leukemia was transplantable with a shorter latency than in the primary transplant setting.
To characterize the tumors at the molecular level, 5 samples from each of the 3 fusions underwent array comparative genomic hybridization, transcriptome, and whole exome sequencing. Samples demonstrated a small number of cooperating mutations with 1.5 copy number alterations (range 0-6) and 6.4 single nucleotide variations (range 2-13) per case. Overall, cases carried an average of 7.9 mutations (range 2-14). Despite the low number of lesions, recurrently mutated genes were identified. These include activating mutations in Flt3, Kras, and cMet, as well as loss of function mutations in the tumor suppressors Phactr4, Wt1, and Tet2. A comparison between fusion subtypes did not reveal any statistically significant differences, although there was a trend towards a greater number of mutations in the GATA2-HOXA9 cohort. Transcriptome sequencing of cohorts, along with normal hematopoietic progenitor subsets, confirmed unique gene expression patterns between each of the fusions. Consistent with immunophenotyping, MN1-FLI1 demonstrated enrichment of the MEP signature while NIPBL-HOXB9 and GATA2-HOXA9 were enriched for CMP and monocyte precursor signatures respectively. ChIP-seq analysis of each of the fusions is underway to definitively identify the genomic targets whose expression is directly altered by their binding.
A common characteristic between all fusions is the presence of protein interaction domains contributed by the N term partner, and DNA binding domains contributed by the C term partner. To determine if these fusions have a novel gain of function distinct from their independent counterparts, we introduced each partner gene into murine bone marrow cells for transplantation experiments. As previously described, introduction of MN1 into hematopoietic cells led to a highly penetrant leukemia. In contrast, HOXA9, HOXB9, and FLI1 all had >75% disease free survival with few myeloid leukemias resulting from their over expression, while GATA2 failed to induce any disease at all. NIPBL’s size precluded transplant assays. Therefore, to evaluate its contribution we introduced a point mutation previously shown to disrupt binding of NIPBL to the cohesion component MAU2. This alteration abrogated the ability of the fusion to induce leukemia in our transplant model, demonstrating the importance of this interaction in the pathogenesis of disease. In conclusion, our data confirms a pathogenic role for GATA2-HOXA9, MN1-FLI1, and NIPBL-HOXB9 in AMKL. Further studies delineating the cooperating mutations required for CBFA2T3-GLIS2 are indicated.
No relevant conflicts of interest to declare.
We have previously defined the promoter of human urokinase‐type plasminogen activator receptor (uPAR) gene in a 188‐bp fragment between bases −141 and +47 relative to the translation start site. ...Here, we report that a novel nuclear factor‐kappaB (NF‐κB)‐like sequence (5′‐GGGAGGAGTC‐3′) at −45 is located in the uPAR promoter and one of the two DNase I‐protected regions, region I between bases −51 and −30. This NF‐κB‐like motif differs at positions 7–9 from the decameric consensus sequences of NF‐κB (5′‐GGGRNNYYCC‐3′ where R indicates A or G, Y indicates C or T, and N indicates any nucleotide) and at positions 1 and 7–9 from the κB‐like motifs (5′‐HGGARNYYCC‐3′ where H indicates A, C, or T, R indicates A or G, Y indicates C or T, and N indicates any nucleotide). Nuclear extracts from HCT116 cells contain proteins that specifically bind to the NF‐κB‐like site at position −45. Mutation of the NF‐κB‐like motif decreased the binding of transcription factor NF‐κB and reduced the uPAR promoter activity in comparison with the wild‐type sequences. Co‐transfection with a dominant negative I‐κB kinase‐2 expression vector reduced uPAR promoter activity by 65–75%. These results demonstrate that a previously uncharacterized NF‐κB motif is required for uPAR promoter activity.
▪
Pathologic germ line mutations that predispose patients to cancer are estimated to occur in 4-30% of all pediatric oncology cases. In addition to leukemia specific familial predisposition ...syndromes, children with rare constitutional syndromes, heterogeneous dysmorphic syndromes, and multiple-cancer hereditary predisposition syndromes are all at an increased risk for hematologic malignancies. However, to date no genome-wide analysis has been done to define the range of germ line mutations that occur in pediatric patients with hematological malignancies.
To determine the frequency of pediatric cancer patients that have germ line variants of pathological significance in genes that predisposed to cancer, we analyzed the germ line and tumor DNA from 1120 pediatric cancer patients that were enrolled in the St. Jude – Washington University Pediatric Cancer Genome Project (PCGP). Samples were analyzed by whole-genome sequencing (n = 595), whole-exome sequencing (n = 456), or both (n = 69). Single nucleotide variants (SNVs), insertions/deletions (indels), structural variations (SV) and copy number alterations (CNAs) were detected using our analytical pipeline and all single nucleotide polymorphisms (SNPs) previously identified in non-cancer populations were filtered out. Our analysis then focused on the 23 cancer predisposition genes recently recommended for germ line analysis by the American College of Genetics and Genomics, along with an additional 8 genes that have been previously shown to predispose to pediatric cancer at a high penetrance. All variants in these 31 genes were classified as pathologic, likely pathologic, uncertain significance, likely benign, and benign based on literature review and in-silico predictions on the effect of novel mutations. An expanded analysis including a total of 565 genes known to play a role in oncogenesis was also evaluated.
Pathologic or likely pathologic germ line variants in one of the 31 genes were detected in 8% (90/1120) of patients, including: 16% (46/287) of patients with solid tumors, 8.6% (21/245) with brain tumors, and 3.9% (23/588) with leukemia. Expanding this analysis to 565 cancer gene resulted in only a slight increase, with a pathologic or likely pathologic variant being detected in 8.6% (97/1120) of patients. The most frequently effected genes included TP53 (n=48), APC (n=7) and BRCA2(n=6). Importantly, in >50% of these patients, analysis of their tumor DNA revealed the absence of a wild type allele for the cancer predisposition gene that was altered in the germ line.
The 588 pediatric patients with leukemia included 116 acute myeloid leukemias (AMLs: FAB M7 n=20; Core Binding Factor leukemias n=86; MLL-R n=10) and 472 acute lymphoblastic leukemias (ALLs: E2A-PBX1 n=53; ERG-R n=39; TEL-AML1 n=53; Hyperdiploid n=69; Hypodiploid n=47; BCR-ABL1 n=40; T-ALL n=32; MLL-R n=40; BCR-ABL-like n=31; and Other n=68). Across this cohort, 3.9% (23/588) of leukemia patients harbored a pathologic germ line mutations in one of the 31 cancer pre-disposing genes. This number increased to 4.6% (27/588; 28 mutations) when the expanded gene list was evaluated. TP53 (n=10) was the most frequently altered germ line gene in pediatric leukemia patients and was found predominantly in low-hypodiploid ALL, as previously reported. Germ line pathologic variants were also identified in KRAS, RUNX1, APC, BRCA2, and RET (2 cases each), and NRAS, SH2B3, BRCA1, MUTYH, PTCH1, SDHA,VHL, and NF2 (1 case each). Although germ line mutations in RUNX1 and SH2B3are typically associated with myeloid neoplasms, we identified these lesions in 3 cases of B lineage ALL suggesting an association with a wider spectrum of leukemia.
In conclusion, a small but significant proportion of pediatric patients with leukemia carry a germ line variant of pathologic significance in a cancer predisposition gene. These results suggest that these germ line lesions likely play a direct role in the pathogenesis of the patient’s presenting leukemia. Moreover, our results suggest that these patients would benefit from future clinical surveillance for the development of a second cancer. Lastly, these data demonstrate the power of comprehensive next generation DNA/RNA sequencing for the identification of pediatric patients who carry a germ line pathologic variant in a cancer predisposition gene.
No relevant conflicts of interest to declare.
Recent genome-wide analyses of acute lymphoblastic leukemia (ALL) have identified genetic alterations targeting the B lymphoid transcription factor PAX5 in over 30% of B-progenitor ALL (Nature 2007; ...446:758; Nature 2008; 453:110). The PAX5 mutations include deletions, focal internal amplification, sequence mutations and translocations that result in PAX5 haploinsufficiency or generate PAX5 mutants with impaired DNA-binding or transactivating activitiy. In almost all B-ALL cases, the mutations are predicted to result in attenuation, but not complete abrogation of PAX5 activity, suggesting that PAX5 is a haploinsufficient tumor suppressor in B-ALL. To test this hypothesis, we have performed mutagenesis screens of mice heterozygous for a Pax5 null allele (Pax5+/−; Cell 1994; 79:901). In a chemical mutagenesis screen, thymectomized C57BL6/Sv129 Pax5+/− (N=25) and wild type (Pax5+/+; N=20) mice received a single 100mg/kg dose of the alkylating agent N-ethyl-S-nitrosourea (ENU) at 4–5 weeks of age. Pax5+/− animals exhibited a markedly increased frequency of leukemia in comparison to wild type animals. After one year, 24 of 25 Pax5+/−animals developed leukemia (median latency 246 days), compared to 3 of 20 Pax5+/+ animals (P<0.0001, Fig. 1). Furthermore, 20 of 22 evaluable Pax5+/− tumors were of B-progenitor cell lineage, in contrast to the Pax5+/+ tumors which were either myeloid (N=2) or T-lymphoid (N=1). Moreover, the Pax5+/−tumors exhibited a range of maturation from pro-B (B220+Cd19-) to pre-B (B220+Cd19+), suggesting the acquisition of additional lesions targeting the B-cell developmental pathway in the less mature tumors. In a complementary retroviral mutagenesis study, thymectomized Pax5+/−(N=32) and Pax5+/+ (N=47) mice received a single dose of Moloney murine leukemia virus at 1–2 days after birth. Pax5+/− mice displayed a higher penetrance and reduced latency of leukemia than Pax5+/+ mice (97% of Pax5+/− mice developed leukemia with median latency of 192 days, v. 66% of Pax5+/+ mice, latency 299 days, P<0.0001). Again, a significantly higher proportion of Pax5+/− tumors (23 of 29 evaluable) were B-progenitor cell lineage than the Pax5+/+ tumors (12 of 26; P=0.01; Fig. 2), and the B-lineage tumors exhibited a spectrum of maturation from pro- to pre-B cell. Cloning and capillary sequencing of retroviral integration sites identified recurring insertions targeting the DNA polymerase gene Poln, the tumor suppressor Wwox, the tyrosine phosphatase Ptprc, the cyclin gene Ccnd3, and orthologs of genes targeted by DNA copy number alterations in human B-ALL including Tbl1xr1. Together, these data indicate that a decrease in the normal transcriptional level of Pax5 directly contributes to the development of B-progenitor ALL, consistent with Pax5 functioning as a haploinsufficient tumor suppressor. Next-generation sequencing to comprehensively identify all genes targeted by retroviral insertions, coupled with genome-wide copy number analysis, are in progress and should provide invaluable information on the range of mutations that cooperate with Pax5 haploinsufficincy to induce ALL.
Display omitted Display omitted
PDF
